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TCR Monday Notes
I was suppose to post this Friday night, but life got in the way the rest of the weekend...
- Arizona Phil does what only he can do...an amazing retrospective on how the 1969 Cubs were built and his memories of the season.
- Ryan Theriot is losing fans in the Cubs organization apparently by sticking to his $3.4M demands. Bruce Miles on the radio and Bruce Levine both saying that an arbitration hearing looks inevitable. It would be the first arbitration hearing in 17 years between the Cubs and a player (Mark Grace).
- Orlando Cabrera signs with the Reds on a 3 million deal for one year with a $1M buyout on a $3M mutual option in 2011. They then acquired Aaron Miles from the A's for Wily Taveras and Adam Rosales. As long as they don't land Johny Damon...
(Interesting twist to this story, A's DFA Taveras immediately. Also, the A's are sending a PTBNL to the Reds as part of the deal).
- Reed Johnson was offered $800K by the Los Angeles Dodgers.
- Wezen Ball relates some interesting press quotes when Andre Dawson signed with the Cubs in 1987. There's been a bit of myth perpetuated that Dawson was dead set to
play for the Cubs at any cost. But what really happened was that he was a victim of
collusion by the owners and the blank check story was a desperate plea to find a team for '87. To his credit though, he was a man of his word and certainly didn't sulk over the low-ball contract offer.
"I didn't think they'd make an offer, because I thought they didn't want to be the first ones to sign a free agent," Dawson said yesterday. "But since they did, I'm man enough to stick by it.
I know I'll miss Montreal . . . but you gotta do what you gotta do. I don't want to say a move was overdue, but with the problems I had the last couple years . . . I had the same salary the last six years, and then they offer me a pay cut . . .
It got so that towards the end of the season some of my own teammates told me it would be best that I make a change."
- An interview with an author of a book on how the Cubs may have thrown the 1918 World Series.
- The Xavier Nady deal was made official Friday. The Cubs will be cautious with Nady in March and April though and he probably won't see much time in the field to start the season, much like Tyler Colvin did at the beginning of last season after his Tommy John surgery. I do wonder what that will do to the bench since it means Fukudome won't have a platoon partner. They could possibly put Baker out there, but that means either Fontenot plays against a lefty or Blanco. They could also carry an extra bat at least for the first two weeks as a 5th starter isn't due until April 18th.
Dr. Hecht has some good info on Nady's surgery and expected recovery in the comments.
- Jayson Stark says the Cubs prefer to sign a reliever such as Chan Ho Park or Kiki Calero, otherwise a trade with the Blue Jays for Jason Frasor or Scott Downs is a possibility.
- Jerry Crasnick tweets that Jermaine Dye essentially turned down the offer that Xavier Nady accepted.
- Today in Get off my Lawn with your Baseball Stats Articles.
Re: TCR Monday Notes
Sunday, April 18, vs. Houston is when we'll need the fifth starter.
Re: TCR Monday Notes
Reds-"As long as they don't land Johny Damon..."
They could land Pujlos, ain't going to matter.
Re: TCR Monday Notes
The article on defensive metrics was pretty good. It would have been better if his shining examples of how to do it right weren't also organizations that value OBP and defense...
I do think that by stacking these defensive improvements next to eachother (CF-LF and 3B-SS) the sum is going to be less than the totals of the parts for the Sox.
Re: TCR Monday Notes
It certainly attempts to address some of the concerns. I would actually like to see fielding preference worked in there, somehow. Since the corner guys get and pitchers shot at the ball before the Middle infielders it seems like that should be incorporated into any model. I'll have to encourage them to look at it over there.
Re: TCR Monday Notes
If this goes to arbitration, Theriot will lose $$ as well as fans. I like Theriot in that he plays hard and gives all he's got. I just don't see him as being worth $3.4 million. I hope both sides will come to an agreement for something under $3 mil. If not, I like the Cubs chances in this one.
Re: Theriot
If I'm Ryan Theriot, I agrue that I've put up numbers very similar to Phil Rizzuto at this age. And he's in the Hall of Fame.
Re: Phil Rizzuto may not qualify as a contemporary
Theriot should counter that Rizzuto was underappreciated by his contemporaries as well. He never got more than 38% of the BBWAA vote. Didn't get in until 1994 when his brilliance became clearer, 38 years after he retired.
Don't let those bastards underpay me, I'm HoF material!
Re: TCR Monday Notes
I was joking, but the arbitration criteria is interesting.
e.g., What are the salaries of comparable players?
~edit~ oops, I see we have one, Stephen Drew.
Re: TCR Monday Notes
Any of the players special accomplishments IE Golden Gloves, All Star appearance
Golden gloves? The player has to know how to box?
Re: TCR Monday Notes
When asking for money, it never hurts that the other party thinks you know how to throw a mean hook.
Re: TCR Monday Notes
Interesting. Also, this:
"The Players contribution to the team in terms of leadership as well as performance"
I had no idea about that clause. How do you quantify leadership in specific dollars and cents? Does scrappiness count?
Re: TCR Monday Notes
More like "Yakety Sax", better known as the Benny Hill theme.
Re: TCR Monday Notes
Oh man, I would pay money to watch Theriot baserunning gaffe's to the benny hill theme. That and caught stealing.
Re: A's DFA Taveras right after making trade.
"be pretty amusing if Reds then DFA Aaron Miles..."
Over Dusty's dead body.
Re: TCR Monday Notes
I like Dana Eveland. His big league numbers are pretty awful, but he's still just a 26 year old lefty with some good stuff.
Had some injury issues throughout his AA and AAA years. I don't think this team has the space to let another youngster figure it out, but somebody just might get him for a steal.
Re: TCR Monday Notes
I was looking at Michael "In Lou's Doghouse and GONE for No Apparent Reason" Wuertz's Baseball Reference page. And lo and behold the pitcher most similar to him... Kiko Calero.
Oh the irony!
Re: TCR Monday Notes
Re: TCR Monday Notes
Let Colvin hit in Iowa until we find significant playing time for him or can get value for him in a trade.
I'd probably prefer Taveras. I don't have any faith that Fuld can stay healthy and Taveras is a better defender and probably a better base stealer, and can get hits out of sac bunts if his legs are right.
Tevaras is a month younger than Fuld, too.
Re: TCR Monday Notes
Re: TCR Monday Notes
Costs being equal, Taveras just seems like a better fit for the team and the role.
But he'll probably be looking for at least a 4th OF job, though I guess there aren't many of those left now.
Re: TCR Monday Notes
He wouldn't exactly be stuck behind Ruth,Mantle and Williams with the Cubs.
Re: TCR Monday Notes
agree about taking a flyer on taveras.
Re: TCR Monday Notes
Have read 'The Original Curse' based on the premise that the 1918 WS was crooked. It's well-written & makes a good circumstantial case that the Cubs were screwed long before the billy goat was booted from WF. It would make a good read between now & the arrival of P's & C's at Phil's place...
Re: TCR Monday Notes
Cubs sniffing around Kris Benson, according to the latest rumors from The Pope over at Foxsports.com
Re: TCR Monday Notes
They are probably just sniffing around his wife, and he happens to be in the same place... Yikes!!
Recent comments
it's day old news, and it's got nothing to do with the cubs, but ichiro signed a $2m deal with MIA (with a $2m option for 2017).
neat. 41 years old and damn close to 3000 hits.
also, rain delays suck.
take that giants
I think that if a team objects to the 1-game wildcard playin game so much, they could just win the pennant and avoid themselves the trouble.
Per Jesse Sanchez at mlb.com, Cubs reportedly have signed 20-year old Cuban OF Eddy Julio Martinez for $3M bonus.
BLOCK: Of course any advantage is an advantage. An MLB, NBA, or NHL team getting the extra game at home in a seven game series is an advantage, I just don't think it is enough of an advantage for winning a division and/or having the best record in a conference or league over the course of an 82-game season (NBA and NHL) or 162 game series (MLB).
TEX takes the opening game from TOR (@TOR) 5-3.
TOR lost bautista + donaldson in-game due to injuries...TEX lost beltre...dunno if any will be lingering issues leading to missed games.
Ride the Kid Magic! Schwarber hadn't homered in a long time before last night.
Greg Maddux was 8-18 in his rookie season. Kyle has the 8 wins down pat.
Think Baby Maddux.
Prof. Harold Hill's THINK system at work.
Kyle is on the far left.
I support this. Hendricks has not only looked better lately but seems to start struggling after a few innings which is better than the 1st in the playoffs.
Just tweeted via Jesse Rogers: Hendricks starting Game 2. Wow. Just wow.
That was good!
Well said. On one hand, I thought the HBP was a bad baseball play -- down 4 runs, put a runner on for a red-hot Fowler. On the other hand, they needed to do something -- I hadn't thought about the warning/pitching inside point. Is Hurdle that smart? He does not strike me that way. By the way -- not clear which fan base you are referring to in your "first" 3rd point.
My unsolicited opinions on topics covered in this thread:
1. I hate the fact that after 162 games, a team could be out after 1 game. However, I think the system is pretty close to perfect right now. 2 of 3 isn't feasible unless they shorten the regular season, and it ices the division winners for way too long. This creates excitement, and rewards the division winners.
Personally, I think the game could have had a very different look had the Pirates held onto the ball and tagged Fowler out on the steal in the first. Cole was clearly frazzled, but if they took that runner off the base, it could have relaxed him a lot.
Football games are played once a week. There are 16 games a year. I'm not even remotely following at all how you can compare the two leagues and playoff systems. It is physically impossible to play a home and away series. The idea of not having any road games in baseball playoffs is certainly a head scratcher.
How is not having the first and last game at home a benefit for the division winners and team with the best record? How is it not an incentive to win the division when a WC team has to blow their top pitcher?
Call me lost.
Two 97+ win teams in a do-or-die, great bullpens, overpowering starters, plenty of pop--hard to believe that game wouldn't be tense. A 4-0 lead is not a blowout, especially in that situation and with the Cubs' young bullpen. Not only would a defensive play here or there make a difference, but you get the win there also on the home plate umps strike zone (generous strike calls for Arrieta, including a couple Ks), and on Schwarber sitting on the right pitch at the right time.


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This item sold on May 12, 2012.
Exquisite bracelet from the 20s-30s featuring sapphire blue faceted crystal stones set in sterling silver with a Greek key motif. Marked STERLING on the female end of clasp. Very good condition, the clasp works properly and there is mild overall wear. 7" by 3/16".
Art Deco Bracelet Sterling Silver Sapphire Blue Glass Crystals Wedding Jewelry
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Savory Pear Stuffing
Spruce up packaged cornbread stuffing mix with Bartlett pears, green onions, and fresh parsley.
Total Time:
Level: Easy
Serves: 8
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- 1 tbsp. olive oil
- 2 stalk celery
- 2 large ripe Bartlett pears
- 4 green onions
- ½ c. fresh parsley leaves
- 3 c. water
- 1 package cornbread stuffing mix
- Preheat oven to 425 degrees F. In 4-quart saucepan, heat oil on medium-high until hot. Add celery and cook 5 minutes, stirring occasionally.
- Meanwhile, cut pears into 1/2-inch chunks, slice green onions, and chop parsley.
- Add water to celery in saucepot; cover and heat to boiling on high. Remove from heat; stir in stuffing mix, pears, green onions, and parsley until combined.
- Spoon stuffing into shallow 3- to 3 1/2-quart casserole; cover with foil. Bake 20 minutes; uncover and bake 10 minutes longer.
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New York City Kids Visitors Guide
Mommy Poppins is the ultimate insider's guide to NYC for local families, but that also makes us an awesome resource for travelers. This guide will help you plan your trip to enjoy New York City like the natives do.
You'll want to hit the big tourist attractions, sure—see our restaurant suggestions for the best local food options nearby—but the real way to experience New York City is to explore different neighborhoods. Check out our neighborhood guides for itineraries that will have you exploring Uptown & Downtown Manhattan, Brooklyn, the Bronx, Queens, or Staten Island like a local. And we'll tell you how to build some fun play into your day so the kids don't burn out.
Make sure to check our Event Calendar to see what's going on while you're here and our Museum Guide for current exhibit reviews. Even better, subscribe to our newsletters so you can plan your visit and stay up-to-date on the coolest happenings. Check out our seasonal guides for the best things to do if you're visiting in the Winter, Spring, Summer, Fall or around the Holidays. And, to really do NYC like a local, try tackling our list of 100 Things to Do in New York City with Kids Before They Grow Up.
Familly-Friendly Hotels
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Few things will excite a kid more on a hike than the sudden roar of rushing water. They'll race toward the falls, eager to toss in a stick to see where it'll go, mesmerized by the crashing water. But really, encountering a waterfall deep in the woods is enchanting at any age.
Families can... read more
There's something about the fall season that makes us antsy for a day trip out of the city with the kids to enjoy the changing leaves with a hike in cooler... read more
We have been telling you about lots of fun farm trips to take this harvest season, most in the tri-state area that are a short drive, train trip or subway... read more
There's still plenty of time this summer to sneak away from the sticky city heat for a little weekend trip. A relaxing family jaunt to coastal Maine, a mountain town in Vermont, the sun and sand at the Jersey shore are all... read more
Car time has become synonymous with screen time. It's just so easy to keep the kids occupied in the backseat with a smartphone or tablet, that the "I'm boreds!" and "Are we there yets?" of our childhoods almost seem like a thing of the past. But I also remember the fun of long car rides: talking... read more


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AAPL Stock: 109.5 ( -1.28 )
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Rice University today used the International Solid-State Circuits Conference to reveal that it has tested a real-world example of a processor founded on probability math. Called a PCMOS (probability-based complementary metal-oxide semiconductor), the chip abandons the either/or Boolean logic of all current processors in favor of calculations that rely on the most likely answer in most cases. By avoiding a reliance on getting an exact answer when unnecessary, the CPU uses just a fraction of the power to accomplish the same work as today's chips; the example chip's voltage is dropped to where it consumes 30 times less power than an equivalent, ordinary CMOS processor.
Mobis Technology has released the latest in its line of mobile stands, Just Mobile Xtand Pro for the Mac. The new mobile stand is created completely out of aluminum (to help match the metal finish of the MacBook) and is made to fit any sized Mac. Just Mobile Xtand Pro comes complete with an adjustable bar that provides height-customization capabilities to help bring the Mac screen up to eye level, and includes rubber pads made for preventing the laptop from falling off.
Innovative Language Learning has released Pocket Italian and language learning apps in eight other languages. The apps are also available for Arabic, French, Italian, Japanese, Korean, and Russian and will be available for English, German, and Spanish. They will be offered at four different levels of aptitude and will all be made available for purchase on iTunes during the month of February. The apps work using pre-recorded lessons and dialogues, self-recordings and dozens of pages of text-book style lesson notes with hours of audio, including compact versions of the company's educational podcasts.
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Deere & Co.'s board of directors approved an increase of the company's share buyback program by $8 billion.
The company also declared a quarterly dividend of 51 cents per share, which will be paid on Feb. 3 to shareholders of record on Dec. 31.
Deere, which is based in Moline, Ill., makes tractors, plows and other gear used by farmers.
Through its share buyback program, the company says it has already repurchased about $11 billion of common stock since 2004.
Deere shares are up $2.99, or 3.6 percent, to $85.70 in morning trading Wednesday.


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Interested in Napa?
We'll send you updates with the latest deals, reviews and articles for Napa each week.
You may also be interested in these hotels within five miles of Napa:
Topics include Transportation, United States: For Foreign Visitors & more!
Although a visit to Napa conjures up images of all kinds of adult-centered activities, the area is remarkably child friendly. There are a variety of activities designed with children in mind.
Some experts suggest staying in Calistoga, just to the north of the town of Napa. From there, you can visit many child-friendly places like the Petrified Forest (at 4100 Petrified Forest Road in Calistoga), which features a variety of petrified trees, some as long as 65 feet. These petrified trees aren't as colorful as the ones found in Arizona, but children will likely marvel at their amazing size and age. there is also an "old faithful" geyser in Calistoga.
If you want to add some wine tasting to the family weekend head for Sterling Vineyards, which it set atop a hill. You get to the tasting room via a tram ride, sure to entertain the kids. Once in the tasting room, you can enjoy the variety of wines (which include Pinot Gris, Cabernet Sauvignon, Syrah, Chardonnay and more), while the children sip sparkling juice.
Or take the children for an exciting castle tour at Castello di Amarosa. They have young adult tours for children 5 to age 20.
If your kids are at least 90 pounds - you can all go on a Segway tour in Napa or Yountville - or rent them by the hour.
For younger kids try Scientopia Discovery Center in the city of Napa. A great place for kids to run around, interact wih exhibits, play with toys or dress up. It has lots of educational areas and tons of arts and crafts.
Close to Napa is the Jelly Belly factory in Fairfield, Mrs. Grossman's Sticker Factory in Petaluma (tours are $3 each), and Six Flags Discovery Kingdom in Vallejo.
If you want to enjoy a quiet dinner out, ask the proprietor of your bed and breakfast or the concierge at your hotel for a babysitting recommendation. There are a variety of agencies in the Napa area that provide licensed and bonded nannies and babysitters who will come right to your hotel or B & B room to watch your children while you head out for an evening.


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2 of 3 people found the following review helpful
Defective Issues - Not for you if you are SERIOUS about your turtles condition,
This review is from: Tetra 26445 Aquatic Reptile Heater, 100 Watt (Misc.)
I had bought this product almost 2 years ago and it worked for a few months until the light stopped turning on when it needed to warm up again. If I unplugged it and plugged it back in it would heat up for a minute and then shut off again. I used a regular glass heater instead and my turtle thankfully did not break it. Later I lost that heater when I moved into my new place and bought this product a second time as I figured it would save me some grief while I was away, and to my dismay it stopped working the day I bought it. I am not even bothering with service or returning it, normally Tetra is completely awesome with their products, but it's unfortunate they do not address this issue. I would suggest to anyone who is SERIOUS about taking care of any turtles that you invest in a better heating device (eheim has some I believe that are expensive but still well worth the money in the long run as they are digital and reliable) You can also get a regular glass heater and make-shift an outer casing for it, or even find a better heater than this one and put the plastic around it from what you got from this product. Essentially the idea is fantastic: my turtle only accidentally opened the plastic casing when she was very little and kept swimming on top of it, but since then she hasn't gotten the cover off (not that the product even works anymore) so the design works, it's the product's main feature that does not: the heating. It's cheaper to just make your own casing for a regular heater and check the tempurature every day before and after you get home and make sure it's the right temp. I've come accustomed to warming my room to the right tempurature so that my tank is appropriately heated (room temp = around 70-75 degrees in the water for me)
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by Steve Haruch
As Betsy mentioned a while back, actor George Takei has lent his name to the fight against Stacey Campfield's embarrassingly dumb "Don't Say Gay" bill. Now he's got a video and some all-important swag to to along with his "It's OK to be Takei" campaign. Maybe basketball players can start using "Takei" in their trash-talk, too:
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Login or Sign Up now to post a comment!2 years agoDo you suppose you are blessed when you have male/male sex in Israel? The ruins wouldn't ruin my sex. Remember to swallow his CUM. It's a sin to spit out his load.2 years ago2 years ago6 months agoThis is a 3some so what we have is FAGGOT Porn video from the gutter(Israelis) and it's due mainly because of the guy wearing the necklace who sucks the cocks and then gets fucked by both guys at the same time---double penetration. Not in my plus column. Using condoms was one good thing.1 month ago3 months agoNot sure why they left the ruins to go fuck in the woods. Sucking and fucking and cumming in 2,000+ year old ruins would be kinda neat I think!10 months ago10 months ago10 months ago10 months ago1 year ago[[dateAdd]][[commentMessage]]0Flag This Video
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Rate Dr. Kevin MacDonald
Dr. Kevin MacDonald's Ratings
Being an out of town patient, I had explained our situation and requested to have our daughters consult and procedure completed in one visit and had requested to be put on a cancellation list. I was pleasantly surprised when we quickly got a cancellation appointment and within hours were off to Saint John. We were greeted by a very friendly and professional staff. Our 12 year old daughter had a frenectomy performed and was feeling faint several times after the procedure. She was tended to thoroughly with oxygen, cold clothes and even a juice box to make her feel better. When Dr. MacDonald was made aware of her condition, he tended to her personally. Staying with us until she felt better. Giving us literature on post surgery protocol and even including his cell phone should there be any area of concern to contact him. Once our daughter was starting to feel better, Dr. MacDonald even walked her by hand to the car to ensure she was going to be ok. I was so impressed by his caring approach, professional and friendly staff. This was a positive experience for our daughter which she will always remember. As well worth mentioning after her gum had healed, she looked in the mirror and said "Perfection Mom, I knew we picked the right doctor". That says it all.
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Several months ago, I had a tissue graph completed by Dr. MacDonald. I have a heart condition and Dr. MacDonald was very careful to ensure all necessary precautions were taken during my procedure (including having a nurse present). Everything was well explained and you can tell he is confident and skilled.
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Great overall experience! I had my frenum cut to prevent my front teeth from spreading, and Dr MacDonald was very informative and efficient. Minimal to no pain!
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he is a wonderful dental surgen. He explains every thing he is doing during a proceedure very carteful and gentle when he works on your mouth. I would highly recommend him to anyone
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In the past 6 months I have had several procedures completed by Dr. MacDonald (dental implant, gingivectomies, crown lengthening, and a gum graft). All work was pain free without any complications. I'm very satisfied with the results. I'm a person who typically asks a lot of questions prior to a procedure and I have never felt rushed. Dr. MacDonald always takes the time to inform you about the procedure. He is an excellent surgeon who I highly recommend for any type of gum surgery, I wouldn't go anywhere else!
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Author contributions: P.Y.T.: conception and design, conducted all experiments, collection and/or assembly of data, data analysis and interpretation, provision of study material, and manuscript writing; P.S.K.: conception and design, collection and/or assembly of data, data analysis and interpretation, provision of study material, financial support, and manuscript writing; N.V.: conception and design, provision of study material in the form of the hESC retroviral library, and manuscript writing.
Cancer Stem Cells
Identification of DPPA4 and DPPA2 as a novel family of pluripotency-related oncogenes
Article first published online: 15 NOV 2013
DOI: 10.1002/stem.1526
Copyright © 2013 AlphaMed Press
Volume 31, Issue 11, pages 2330–2342, November 2013
Additional Information
How to Cite
Tung, P.-Y., Varlakhanova, N. V. and Knoepfler, P. S. (2013), Identification of DPPA4 and DPPA2 as a novel family of pluripotency-related oncogenes. STEM CELLS, 31: 2330–2342. doi: 10.1002/stem.1526
Publication History
- Issue published online: 15 NOV 2013
- Article first published online: 15 NOV 2013
- Accepted manuscript online: 20 AUG 2013 05:48AM EST
- Manuscript Revised: 7 JUL 2013
- Manuscript Accepted: 7 JUL 2013
- Manuscript Received: 15 FEB 2013
- Cancer;
- Cancer stem cells;
- Cell cycle;
- Pluripotent stem cells;
- Embryonic stem cells;
- Pluripotency-related oncogenes
In order to identify novel pluripotency-related oncogenes, an expression screen for oncogenic foci-inducing genes within a retroviral human embryonic stem cell cDNA library was conducted. From this screen, we identified not only known oncogenes but also intriguingly the key pluripotency factor, DPPA4 (developmental pluripotency-associated four) that encodes a DNA binding SAP domain-containing protein. DPPA4 has not been previously identified as an oncogene but is highly expressed in embryonal carcinomas, pluripotent germ cell tumors, and other cancers. DPPA4 is also mutated in some cancers. In direct transformation assays, we validated that DPPA4 is an oncogene in both mouse 3T3 cells and immortalized human dermal fibroblasts. Overexpression of DPPA4 generates oncogenic foci (sarcoma cells) and causes anchorage-independent growth. The in vitro transformed cells also give rise to tumors in immunodeficient mice. Furthermore, functional analyses indicate that both the DNA-binding SAP domain and the histone-binding C-terminal domain are critical for the oncogenic transformation activity of DPPA4. Downregulation of DPPA4 in E14 mouse embryonic stem cells and P19 mouse embryonic carcinoma cells causes decreased cell proliferation in each case. In addition, DPPA4 overexpression induces cell proliferation through genes related to regulation of G1/S transition. Interestingly, we observed similar findings for family member DPPA2. Thus, we have identified a new family of pluripotency-related oncogenes consisting of DPPA2 and DPPA4. Our findings have important implications for stem cell biology and tumorigenesis. Stem Cells 2013;31:2330–2342


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Lymphatic and Angiogenic Candidate Genes Predict theDevelopment of Secondary Lymphedema followingBreast Cancer SurgeryChristine Miaskowski1*, Marylin Dodd1, Steven M. Paul1, Claudia West1, Deborah Hamolsky2,Gary Abrams3, Bruce A. Cooper4, Charles Elboim5, John Neuhaus6, Brian L. Schmidt7, Betty Smoot8,Bradley E. Aouizerat91Department of Physiological Nursing, University of California San Francisco, San Francisco, California, United States of America, 2Department of Physiological Nursing,Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, California, United States of America, 3Department of Neurology,University of California San Francisco, San Francisco, California, United States of America, 4Department of Community Health Systems, University of California SanFrancisco, San Francisco, California, United States of America, 5Redwood Regional Medical Group, Santa Rosa, California, United States of America, 6Department ofEpidemiology and Biostatistics, University of California San Francisco, San Francisco, California, United States of America, 7Department of Oral and Maxillofacial Surgery,New York University, New York, New York, United States of America, 8Department of Physical Therapy and Rehabilitation Science, University of California San Francisco,San Francisco, California, United States of America, 9Department of Physiological Nursing, Institute for Human Genetics, University of California San Francisco, SanFrancisco, California, United States of AmericaAbstractThe purposes of this study were to evaluate for differences in phenotypic and genotypic characteristics in women who didand did not develop lymphedema (LE) following breast cancer treatment. Breast cancer patients completed a number ofself-report questionnaires. LE was evaluated using bioimpedance spectroscopy. Genotyping was done using a customgenotyping array. No differences were found between patients with (n=155) and without LE (n=387) for the majority ofthe demographic and clinical characteristics. Patients with LE had a significantly higher body mass index, more advanceddisease and a higher number of lymph nodes removed. Genetic associations were identified for four genes (i.e., lymphocytecytosolic protein 2 (rs315721), neuropilin-2 (rs849530), protein tyrosine kinase (rs158689), vascular cell adhesion molecule 1(rs3176861)) and three haplotypes (i.e., Forkhead box protein C2 (haplotype A03), neuropilin-2 (haplotype F03), vascularendothelial growth factor-C (haplotype B03)) involved in lymphangiogensis and angiogenesis. These genetic associationssuggest a role for a number of lymphatic and angiogenic genes in the development of LE following breast cancer treatment.Citation: Miaskowski C, Dodd M, Paul SM, West C, Hamolsky D, et al. (2013) Lymphatic and Angiogenic Candidate Genes Predict the Development of SecondaryLymphedema following Breast Cancer Surgery. PLoS ONE 8(4): e60164. doi:10.1371/journal.pone.0060164Editor: Robert Lafrenie, Sudbury Regional Hospital, CanadaReceived February 1, 2013; Accepted February 21, 2013; Published April 16, 2013Copyright: ? 2013 Miaskowski et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Funding: This study was funded by grants from the National Cancer Institute (CA107091 and CA118658) and the National Institute of Nursing Research(NR0101282). Dr. Miaskowski is an American Cancer Society Clinical Research Professor. Dr. Bradley Aouizerat was funded through the National Institutes of Health(NIH) Roadmap for Medical Research Grant (KL2 RR624130). This project was supported by NIH/NCRR UCSF-CTSI Grant Number UL1 RR024131. Its contents aresolely the responsibility of the authors and do not necessarily represent the official views of the NIH. The funders had no role in the study design, data collectionand analysis, decision to publish, or preparation of the manuscript.Competing Interests: The authors have declared that no competing interests exist.* E-mail: firstname.lastname@example.com (LE) following treatment for breast cancer is themost common form of LE in the industrialized world [1]. LE iscaused by a reduction in lymph transport capacity associated withthe cancer and its treatment (e.g., surgery, radiation therapy (RT),chemotherapy (CTX)) and results in the accumulation of protein-rich fluid in the interstitial space. LE results in disfigurement,discomfort, and functional impairments. In addition, LE mayprecipitate cellulitis and lymphangitis [2].The exact prevalence of LE in breast cancer survivors isunknown with estimates ranging from 3% to 87% [3]. This widevariation is due to differences in measurement methods, inconsis-tencies in diagnostic criteria, as well as variations in samplecharacteristics, timing of measurements, and duration of follow-up. However, as noted by Armer [3], if the incidence of LE isconservatively set at 20%, more than 500,000 breast cancersurvivors in the United States are affected by this condition.One of the major goals of LE research is the identification ofwomen at greatest risk for the development of this conditionfollowing breast cancer treatment. Risk factors evaluated in themajority of studies done to date focused on patient, disease, andtreatment characteristics. However, in four large scale prospectivecohort studies [4–7], the factors associated with increased risk forLE were not concordant. In a study of 1,287 women (104 werediagnosed with LE) [5], risk factors for LE included: higher stageof disease, increased number of lymph nodes removed, removal oftumor positive nodes, receipt of adjuvant CTX, higher body massindex (BMI), and poorer health status. In a second study of 997women (133 had LE) [4], risk factors for LE included: beingAfrican American, being better educated, higher stage of disease,and removal of at least one lymph node. In a population-basedsample of 631 women (237 had LE) [6], hazard ratios for thePLOS ONE | www.plosone.org1 April 2013 | Volume 8 | Issue 4 | e60164
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development of LE were increased following axillary lymph nodedissection (ALND) and receipt of CTX. Finally, in a sample ofbreast cancer patients (n=2,431) from the Women's HealthyEating and Living Well Study [7], women with LE (28.5% of thesample) were diagnosed at a younger age, had a higher BMI, had alarger tumor size, had more lymph nodes removed, were morelikely to have a mastectomy with radiation therapy (RT) and weremore likely to have CTX. While all four studies evaluated a largenumber of risk factors, the diagnosis of LE relied on patient self-report [5–7] or medical record data [4].While the phenotypic characterization of risk for LE followingbreast cancer treatment warrants additional investigation, recentevidence suggests that some of the variation in the occurrence andtime to onset of LE may be related to genomic determinants [8,9].These two studies evaluated candidate genes that are known toplay a role in lymphatic development [10–15] or were identified instudies of primary LE which are Mendelian inherited disorders. Ina study that evaluated hepatocyte growth factor (HGF) and thehigh affinity hepatocyte growth factor receptor (MET) in 59women with breast cancer related LE [9], mutations leading totruncation or missense changes in evolutionarily conservedresidues of HGF and MET were identified. In a case controlstudy of 188 women (80 diagnosed with LE) [8], mutations inGJC2 that encodes for connexin 47 were identified in women withLE. Taken together, these findings suggest that additional studiesare warranted to determine the phenotypic and genotypic factorsassociated with increased risk for LE following breast cancertreatment. Therefore, the purposes of this study were to evaluatefor differences in phenotypic and genotypic characteristics inwomen who did and did not develop LE following breast cancertreatment.MethodsStudy Samples and ProceduresDemographic, clinical, and genomic data from a cross-sectionalstudy (i.e., LE Study (NR0101282)) and a longitudinal study (i.e.,Breast Symptoms Study (CA107091 and CA118658)) werecombined for these analyses. Both studies used the same subjectiveand objective measures. Both studies were approved by the UCSFCommittee on Human Research and the CTSI Clinical ResearchCenter Advisory Committee.LE Study.The LE study used a cross-sectional design toevaluate for differences in phenotypic and genotypic characteris-tics in women with (n=74) and without LE (n=71). Women whowere $18 years of age, $6 months post-treatment for unilateralbreast cancer, and with or without upper extremity LE wererecruited. Women were excluded for bilateral breast cancer,current upper extremity infection, lymphangitis, preexisting LE,current breast cancer, or contraindications to bioimpedancespectroscopy (BIS) testing. Women were recruited through theNational Lymphedema Network website, San Francisco Bay areahospitals, and breast cancer or LE support groups and conferenc-es. Women were evaluated in the Clinical Research Center atUCSF. After obtaining written informed consent, women com-pleted the study questionnaires. Following the completion of thequestionnaires, the research staff performed the objective mea-surements: height, weight, and BIS. A blood sample was drawn forgenomic analyses.Breast Symptoms Study.The Breast Symptoms Study useda prospective, longitudinal design to evaluate neuropathic painand LE following breast cancer surgery. Women were recruitedfrom Breast Care Centers located in a Comprehensive CancerCenter, two public hospitals, and four community practices.Patients were eligible to participate if they were: adult women($18 years) who would undergo breast cancer surgery on onebreast; able to read, write, and understand English; agreed toparticipate, and gave written informed consent. Patients wereexcluded if they were: having breast cancer surgery on bothbreasts and/or had distant metastasis at the time of diagnosis. Atotal of 516 patients were approached to participate, 410 wereenrolled in the study (response rate 79.4%), and 398 completed thepreoperative assessment. The major reasons for refusal were: toobusy, overwhelmed with the cancer diagnosis, or insufficient timeavailable to do the baseline assessment prior to surgery. During thepatient's preoperative visit, a clinician explained the study,determined the patient's willingness to participate, and introducedthe patient to the research nurse. The research nurse met with thewoman, determined eligibility, and obtained written informedconsent prior to surgery.After obtaining written informed consent, the patient completedthe enrollment questionnaires prior to surgery. Following thecompletion of the questionnaires, the research nurse performedthe objective measurements: height, weight, and BIS. A bloodsample was drawn for genomic analyses. Patients were contactedtwo weeks after surgery to schedule the first post-surgicalappointment. The research nurse met with the patients either intheir home or in the Clinical Research Center at 1, 2, 3, 4, 5, 6, 8,10, and 12 months after surgery. In the second through fifth yearsof the study, patients were seen every four months. During each ofthe study visits, the women completed the study questionnairesand had the objective measures done by the research nurse.Subjective MeasuresA demographic questionnaire was used to obtain informationon age, marital status, education, ethnicity, employment status,living situation, and financial status. Functional status wasevaluated using the Karnofsky Performance Status (KPS) scalethat has well established validity and reliability [16,17]. Patientsrated their functional status using the KPS scale that ranged from30 (I feel severely disabled and need to be hospitalized) to 100 (Ifeel normal; I have no complaints or symptoms). Patients wereasked to indicate if they exercised on a regular basis (yes/no).Clinical information was obtained from patient interviews andmedical record reviews.Self-Administered Comorbidity Questionnaire (SCQ) is a shortand easily understood instrument that was developed to measurecomorbidity in clinical and health service research settings [18].The questionnaire consists of 13 common medical conditions thatwere simplified into language that could be understood withoutany prior medical knowledge. Patients were asked to indicate ifthey had the condition using a ''yes/no'' format. If they indicatedthat they had a condition, they were asked if they receivedtreatment for it (yes/no; proxy for disease severity) and did it limittheir activities (yes/no; indication of functional limitations).Patients were given the option to add two additional conditionsnot listed on the instrument. For each condition, a patient canreceive a maximum of 3 points. Because there are 13 definedmedical conditions and 2 optional conditions, the maximum scoretotals 45 points if the open-ended items are used and 39 points ifonly the closed-ended items are used. The SCQ has well-established validity and reliability and has been used in studiesof patients with a variety of chronic conditions [18–22].Objective MeasuresBioimpedance Spectroscopy (BIS) of LE.ments, of the affected and unaffected arm, were done using theprocedures described by Cornish and colleagues [23–25]. PatientsBIS measure-Candidate Genes and LymphedemaPLOS ONE | www.plosone.org2 April 2013 | Volume 8 | Issue 4 | e60164
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Table 1. Lymphatic and angiogenic genes and single nucleotide polymorphisms analyzed for lymphedema versus nolymphedema.GeneSNP PositionChr MAFAllelesChi Squarep-valueModelANGPT2 rs291671663446438 .287T.A .380 .827AANGPT2 rs2442468 63513588 .493G.C .073 .964AANGPT2 rs251540963523308 .146T.C .131.937AANGPT2 rs251541363530288 .205T.C 1.464 .481AANGPT2 rs244263663543698 .446T.C .346.841AANGPT2 rs244263163561848 .428G.A .096.953AANGPT2 rs198238663588848 .314G.A .542.762AANGPT2 rs251546263710078 .287G.A 2.660.264AANGPT2 rs699002063736128 .380T.C FE .040DANGPT2 rs251546663736948 .231G.A .230 .892AANGPT2 rs244260863740288 .447A.G .985 .611AANGPT2 rs73470163756558 .329T.C 1.570.456AANGPT2 rs251547763760488 .131C.T .904 .636AANGPT2 rs12674822 63766248 .440G.T .865 .649AANGPT2 rs251548363796778 .353G.C .644.725AANGPT2 rs17552444 63815878 .269A.G .731.694AANGPT2 rs11989215 63833178 .338A.G .371 .831AANGPT2 rs1198924263834288 .478G.A .567.753AANGPT2rs1113703763835908 .326A.C 3.009.222AANGPT2rs1762331363837498 .431C.T .150 .928AANGPT2rs1326902163841718 .270G.T.176 .916AANGPT2rs182337563844068 .349C.G 1.537.464AANGPT2rs13255574 63860858 .204C.T .208 .901AANGPT2rs2922869 63872018 .349A.G 1.543.462AANGPT2 rs251548863904148 .458A.C 2.811 .245AHapA04.119.942HapA06.533.766HapB01.093 .955HapB04 .203.903HapC01 3.938.140HapC042.438.295HapD011.570.456HapD021.087 .581HapD03.797 .671HapE011.816 .403HapE02.682.711HapE03.860 .650HapF01 3.375 .185HapF07.158 .924HapG01 .677.713HapG02 1.392 .499HapG03 .295 .863HapH02 1.523.467HapH03 .202.904FOXC2 rs3422122185157931 16.452T.C 2.830 .243AFOXC2 rs116405908515994516 0.000C.AAll patients were homozygous for CCFOXC2rs1035550 85160208 16.087C.T3.934 .140AHapA012.830.243Candidate Genes and LymphedemaPLOS ONE | www.plosone.org3April 2013 | Volume 8 | Issue 4 | e60164
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Table 1. Cont.GeneSNP Position ChrMAFAlleles Chi Squarep-valueModelHapA03 6.214.045HGF rs5745692811962027.033G.C n/an/an/aHGFrs757830 812003207 .221T.C 1.998.368AHGF rs5745616 812362927 .222G.A 1.315.518ALCP2 rs37891841696116155 .494T.C 2.794 .247ALCP2 rs572192 1696211755 .410A.G FE .047DLCP2 rs10475933 1696228255 .285C.T .409 .815ALCP2 rs23388721696251325 .366A.G 1.492.474ALCP2 rs3157451696302855 .459T.C .431.806ALCP2 rs22711461696349685 .466G.T .119 .942ALCP2 rs2338873 1696412695 .141G.A 3.164 .206ALCP2 rs3157211696476165 .305A.G FE .005DLCP2 rs1826181696556915 .278A.G 4.040 .133ALCP2 rs68667331696558075 .197C.T FE.026DLCP2 rs315730 1696569025 .434A.T .644 .725AHapA03.510.775HapA044.304 .116LYVE1 rs173188581053626311.176T.C .547.761AMETrs714180 1161062387 .488G.A .981.612AMETrs38841 1161071627 .362A.G .0011.000AMETrs39747 1161082757 .441T.C 2.782.249AMET rs38845 1161090387 .456G.A 4.550 .103AMET rs39748 1161146667 .442G.C1.177 .555AMET rs38849 1161197757 .249G.C 2.414.299AMET rs2237710 1161245887 .289T.G 1.666 .435AMET rs38850 1161248857 .246G.A 2.491.288AMET rs117622131161265187 .053G.A 1.434 .488AMET rs22994371161287247 .244G.A 1.244.537AMETrs38857 1161526497 .275C.T 1.954.376AMETrs24021181162158097 .338C.A 1.915 .384AMET rs193686 1162186637 .287T.C 1.569.456AMETrs2023748 1162232587 .447G.A 2.968.227AMET rs1621 1162248427 .318A.G 1.909.385AHapA012.427 .297HapA034.016.134HapB02 2.545 .280HapC023.296.192HapC06 1.704.427HapC07 2.003.367NRP2 rs15175272062525472 .408C.T1.126 .569ANRP2rs6711044 2062529102 .479T.C .413 .813ANRP2 rs1400733 2062531682 .432C.G .329.848ANRP2 rs38065772062543952 .412A.G .291 .865ANRP2 rs8610782062602232 .351A.G 1.320.517ANRP2rs8495302062640492 .482T.G FE.042RNRP2rs9502192062688582 .308G.A 3.601.165ANRP2rs3771052 2062696722 .293G.A 2.566 .272ANRP2 rs8495562062715032.305G.A 1.858.395ANRP2 rs8637072062844282 .445T.G .678.712ACandidate Genes and LymphedemaPLOS ONE | www.plosone.org4 April 2013 | Volume 8 | Issue 4 | e60164
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Table 1. Cont.GeneSNP PositionChr MAFAllelesChi Square p-valueModelNRP2 rs8495252063014862 .466G.A .635 .728ANRP2 rs3771033 2063040552 .326G.A 3.654.161ANRP2 rs8495232063041812 .279C.T 1.299.522ANRP2rs1983343 2063049082 .343A.G 1.258.533ANRP2rs8495842063099612 .318G.T 2.925 .232ANRP2 rs37710212063182332 .405C.T .723.697ANRP2 rs849563 2063187472 .187A.C FE .012RNRP2rs19964122063208302 .473A.G 4.227.121ANRP2rs22411562063232092 .358G.C 1.564.457ANRP2 rs22411552063233112 .358C.T .293.864ANRP2rs3771016 2063237842 .418G.A .005.998ANRP2 rs37710102063318392 .477G.C .219.896ANRP2rs8673442063356602 .346C.T .783.676ANRP2rs3771004 2063391322 .305G.A .006 .997ANRP2 rs168376372063394992 .402G.A .417.812ANRP2 rs168376412063431142 .371G.A FE .009RNRP2rs2241153 2063443682 .383G.T 1.748 .417ANRP2 rs21603282063505822 .374C.T 2.150 .341ANRP2 rs46755422063534922 .213G.C 1.940 .379ANRP2rs109321252063605452 .456G.C 3.922 .141ANRP2 rs37552322063612082 .253A.G 3.096 .213ANRP2 rs15994 2063705422 .377C.G 1.106 .575AHapA01 5.793 .055HapA062.709 .258HapB01 3.427.180HapB04 .631 .730HapB051.340.512HapC01 .163.922HapC02.367 .832HapC03 3.431.180HapD01 .137.934HapD06 .430 .807HapE013.083 .214HapE02.372.830HapE03.006 .997HapF01 8.937.011HapF03 1.574 .455HapF061.426.490PROX1 rs3408742122258791 .499A.G 1.919.383APROX1rs3408392122284431 .483C.T 2.961 .227APROX1 rs7263342122467411 .284C.T .726.696ARORC rs98261500455231 .371A.G .097.953ARORC rs9395951500503121 .381C.A .232.891ARORCrs7540530 1500574821 .466G.A .420 .811ARORC rs112048941500597981 .225G.T3.952 .139AHapA01.420 .811HapA03 .624.732HapA064.005.135SOX17 rs125417425553370710 .210C.T FE .008DCandidate Genes and LymphedemaPLOS ONE | www.plosone.org5April 2013 | Volume 8 | Issue 4 | e60164
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Table 1. Cont.GeneSNP Position ChrMAFAlleles Chi Squarep-valueModelSYKrs1319677 926058209 .484T.C .171 .918ASYK rs1333633 926073939 .416T.C .835.659ASYK rs3789889926111629 .339A.G 2.008 .366ASYK rs290237926144719 .204A.G .034 .983ASYK rs4744505 926198189 .391G.A .140 .932ASYK rs2065583926323839 .151G.C 1.174 .556ASYK rs290213 926356289 .143A.G 1.116 .572ASYKrs1870660 926378269 .163C.G .682 .711ASYKrs1864202926417769 .261C.A 2.573 .276ASYK rs17489214 926429599 .186G.A .342 .843ASYK rs2035073926492409 .328T.C .093 .955ASYK rs11787537926564409 .185G.A .576 .750ASYK rs10993726 926605699 .214C.T .678 .713ASYKrs9695993926635859 .123A.C .742.690ASYK rs290229926742349 .259C.T 3.746 .154ASYKrs10761395 926827189 .300T.C 2.009 .366ASYKrs290254 926917069 .408G.C .078 .962ASYK rs158689926975829 .456A.T FE .039RSYKrs1049164 926980279 .178G.A 2.849 .241AHapA01.204 .903HapA04 .688 .709HapB01.617 .735HapB02 .161 .923HapB03 .029 .985HapC011.608 .448HapD019.769 .008HapD03.093.955HapE01 3.746 .154HapE02.024 .988HapE032.009 .366HapF01 5.000 .082HapF035.254 .072VCAM1 rs14094191009559841 .498T.C 1.095.578AVCAM1 rs31768601009598071 .412A.G .880.644AVCAM1rs3176861 1009599091 .201C.T FE .036DVCAM1 rs39170121009682471 .350T.G 3.381.184AVCAM1 rs31810881009712961 .167C.T 1.520.468AVCAM1rs3176877 1009759831 .396T.A 2.366.306AVCAM1 rs31768791009764151 .061A.G 1.579.454AHapA01 1.579.454HapA05.825 .662HapB01 8.241.016HapB022.366 .306VEGFB rs37414036375610511.433G.A .553 .759AVEGFC rs1485762 1778447254 .310C.T 5.000 .082AVEGFC rs76644131778457014 .206C.T 6.364.041AVEGFC rs68288691778471274 .451C.G .329 .848AVEGFCrs1485766 1778478784 .469A.C .219 .896AVEGFC rs37752021778482054 .494A.G 2.226.329ACandidate Genes and LymphedemaPLOS ONE | www.plosone.org6 April 2013 | Volume 8 | Issue 4 | e60164
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Table 1. Cont.Gene SNPPosition ChrMAF AllelesChi Squarep-valueModelVEGFCrs3775195 1778581044 .238C.A 1.112.573AVEGFCrs37751941778608714 .407C.G .482 .786AVEGFCrs1485765 1778649464 .156A.G 1.453.484AHapA01.297 .862HapA05 6.173 .046HapB012.463 .292HapB037.194 .027HapB04 1.254 .534VEGFDrs6527518 15276100X .435G.T .009 .996AVEGFD rs641868615297927X .329T.C .116 .944AVEGFD rs483093915309204X .324G.A .182 .913AVEGFD rs6632528 15312319X .258T.C .264 .876AHapA01 .001.999HapA02 .474.789HapA03 .325.850VEGFR2 rs12642307556469384 .256T.C 2.524 .283AVEGFR2 rs1531289556499894 .288G.A .111 .946AVEGFR2 rs7671745556515934 .329G.A .557 .757AVEGFR2 rs6828477 556615584 .398T.C .801 .670AVEGFR2rs2168945556622404 .327T.G 1.008 .604AVEGFR2rs1870377556677314 .254T.A 3.543 .170AVEGFR2rs2034965 556725574 .278G.A .923 .630AVEGFR2rs11941492 556729674 .243C.T 2.307 .316AVEGFR2 rs10020464556738274 .330C.T FE .025DVEGFR2rs11133360556775094 .455T.C FE.032RVEGFR2rs1531290 556813194 .437A.G .836 .658AVEGFR2rs12502008556857994 .397G.T 1.751.417AHapA01.386.825HapA02.604 .739HapA03.111 .946HapB023.331 .189HapB03 1.096.578HapB04.716 .699HapC014.155 .125HapC03.278.870HapC047.730 .021VEGFR3 rs2242216 1799740975 .430G.A .137 .934AVEGFR3 rs400330 1799742685 .375T.C .274 .872AVEGFR3 rs307823 1799847145 .271A.G 1.617.446AVEGFR3 rs37971021799877945 .392T.C .255 .880AVEGFR3 rs22909831799915695 .453T.C .708 .702AVEGFR3 rs10085109 1799934105.484C.G n/a n/an/aVEGFR3 rs11748431 1800013475 .237G.A 2.704.259AVEGFR3 rs307814 1800068545 .387C.T .179 .914AHapA01.276 .871HapA03 .137.934HapB02FE .643HapB03 3.693 .158HapB04 .453.797Candidate Genes and LymphedemaPLOS ONE | www.plosone.org7 April 2013 | Volume 8 | Issue 4 | e60164
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were instructed not to exercise or take a sauna within 8 hours ofthe assessment. In addition, they were asked to refrain fromdrinking alcohol for 12 hours prior to the assessment. BISmeasurements were taken using a single channel BIS device (i.e.,SFB7 device; ImpediMed, San Diego, CA in the LE study or theQuantum X Bioelectrical Impedance Device; RJL Systems,Clinton Township, MI in the Breast Symptoms Study). Womenremoved all jewelry and their skin was prepped with an alcoholwipe prior to surface electrode placement. Patients lay supine on amassage table with their arms 30 degrees from the body and legsnot touching for at least 10 minutes prior to the BIS measure-ments. Electrodes were placed on the dorsum of the wristsadjacent to the ulnar styloid process, the dorsum of the hands justproximal to the third metacarpophalangeal joint, anterior to theankle joints between the malleoli, and over the dorsum of the feetover the third metatarsal bone just proximal to the thirdmetatarsophalangeal joint. Two 'measurement' electrodes wereplaced at either end of the 40 cm length over which thecircumference measurements were made and the 'drive' electrodeswere placed 8 to 10 cm distal to these measurement electrodes.These electrode sites were chosen, for the segmental measurementof the arm, in preference to the standard shoulder to wrist sites sothat direct comparisons could be made between the volumesmeasured by the circumference method and by the BIS method.Two readings of resistance were obtained from the affected andunaffected arms and averaged for subsequent analyses.While cases and non-cases of LE were known in the LE study,for the Breast Symptoms Study, LE cases were determined basedon the procedures of Cornish and colleagues [23–25], using all ofthe data obtained from each woman during her participation inthe study. A woman was defined as a LE case if the resistance ratiofor the untreated arm/treated arm prior to surgery was .1.139 or.1.066 for those women who had surgery on the dominant ornondominant side, respectively at any of the BIS assessments.Methods of Analysis for Phenotypic DataData were analyzed using SPSS Version 19 [26]. Descriptivestatistics and frequency distributions were generated on the samplecharacteristics. Independent sample t-tests, Chi-square analyses,and Mann Whitney U tests were done to evaluate for differences indemographic, clinical, and genotypic characteristics betweenpatients with and without LE. Logistic regression analyses wereperformed to evaluate the association between phenotypiccharacteristics and LE group membership.Methods of Analysis for Genomic DataGene Selection.Candidate genes for secondary LE includegenes that cause monogenic (i.e., primary) forms of LE or genesthat cause primary LE in animal models [27–31]. While thegenetic causes of primary LE might be due to ablative mutationsor variations in these candidate genes, secondary LE may becaused by modest functional variations that do not result in criticalloss of gene function. Candidate genes for primary LE includeangiopoeitin-2 (ANGPT2), elastin microfibril interfacer (EMI-LIN1), Forkhead box protein C2 (FOXC2), hepatocye growthfactor (HGF), lymphocyte cytosolic protein 2 (LCP2), lymphaticvessel endothelial hyaluronan receptor 1 (LYVE1, XLKD1),hepatocyte growth factor receptor (MET), neuropilin-2 (NRP2),Prospero-related homeobox 1 (PROX1), ROR orphan receptor C(RORC), SpSRY-box 17 (SOX17), protein tyrosine kinase (SYK),vascular cell adhesion molecule 1 (VCAM1), and vascularendothelial growth factor-B (VEGFB), -C (VEGFC), -D (VEGFD),-receptor 2 (VEGFR2), and -receptor 3 (VEGFR3).Blood collection and genotyping.extracted from archived buffy coats using the PUREGene DNAIsolation System (Invitrogen, Carlsbad, CA). Of the 543 patientsrecruited for this study, DNA was recovered from the archive buffycoat of 407 patients (i.e., 110 with and 297 without LE) whoprovided a blood sample. No differences were found in anydemographic and clinical characteristics between patients who didand did not choose to participate in the studies or between thosepatients who did and did not provide a blood sample for genomicanalyses.Genotyping was performed blinded to LE status and positiveand negative controls were included. DNA was quantitated with aNanodrop Spectrophotometer (ND-1000) and normalized to aconcentration of 50 ng/mL (diluted in 10 mM Tris/1 mMEDTA). Samples were genotyped using the GoldenGate genotyp-ing platform (Illumina, San Diego, CA) and processed accordingto the standard protocol using GenomeStudio (Illumina, SanDiego, CA). Signal intensity profiles and resulting genotype callsfor each single nucleotide polymorphism (SNP) were visuallyinspected by two blinded reviewers. Disagreements were adjudi-cated by a third reviewer.SNP Selection.A combination of tagging SNPs and literaturedriven SNPs (i.e., SNPs reported as being associated with LE) wereselected for analysis. Tagging SNPs were required to be common(i.e., estimated to have a minor allele frequency $.05) in publicdatabases (e.g., HapMap). In order to ensure robust geneticassociation analyses, quality control filtering of SNPs wasperformed. SNPs with call rates of ,95% or Hardy-Weinbergp-values of ,.001 were excluded.As shown in Table 1, a total of 157 SNPs among the 17candidate genes (ANGPT2: 25 SNPs, FOXC2: 3 SNPs; HGF: 3SNPs; LCP2: 11 SNPs; LYVE1: 1 SNP; MET: 15 SNPs; NRP2:32 SNPs; PROX1: 3 SNPs; RORC: 4 SNPs; SOX17:1 SNP;Genomic DNA wasTable 1. Cont.Gene SNPPositionChr MAFAlleles Chi Squarep-valueModelHapC01 .556.757HapC023.163 .206HapC03.490 .783Abbreviations: A= Additive model; ANGPT2 = angiopoeitin-2; Chr = chromosome; D= Dominant model; FOXC2 – forkhead box C2; HGF = hepatocyte growth factor;LCP2 = lymphocyte cytosolic protein 2; LYVE1 = lymphatic vessel endothelial hyaluronan receptor 1 (XLKD1); MAF = minor allele frequency; MET = hepatocytegrowth factor receptor; n/a = not assayed because SNP violated Hardy-Weinberg expectations (p,.001); NRP2 – neuropilin-2; PROX1 = prospero-related homeobox 1;R= Recessive model; RORC = ROR orphan receptor C; SNP = single nucleotide polymorphism; SOX17 = SpSRY-box 17; SYK = protein tyrosine kinase; VCAM1 =vascular cell adhesion molecule 1; VEGFB = vascular endothelial growth factor B; VEGFC = vascular endothelial growth factor C; VEGFD = vascular endothelial growthfactor D; VEGFR2 = vascular endothelial growth factor receptor 2; VEGFR3 = vascular endothelial growth factor receptor 3.doi:10.1371/journal.pone.0060164.t001Candidate Genes and LymphedemaPLOS ONE | www.plosone.org8 April 2013 | Volume 8 | Issue 4 | e60164
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Table 2. Differences in demographic and clinical characteristics between patients with (n=155) and without (n=387)lymphedema.CharacteristicNo LymphedemaLymphedemaStatisticsMean (SD)Mean (SD)Age (years)54.9 (11.1) 56.2 (10.8)NSEducation (years)16.0 (2.7)15.8 (2.8)NSAge at menopause (years)47.8 (7.2)46.7 (9.1)NSBody mass index (kg/m2) 26.1 (5.6)28.2 (6.7)p=.001Karnofsky Performance Status score93.3 (9.7) 91.1 (11.1)p=.028Comorbidity score4.0 (2.9) 4.5 (3.3)NSNumber of nodes removed5.8 (6.3) 10.9 (9.0)p,.0001Number of positive nodes0.7 (1.7)1.7 (3.4)p=.009% (n) % (n)EthnicityWhite 68.8 (265) 72.9 (113)Black 7.5 (29)9.7 (15) NSAsian/Pacific Islander13.0 (50) 7.1 (11)Hispanic/Mixed ethnic background/Other10.6 (41) 10.3 (16)Lives aloneYes23.0 (88) 28.9 (44)NSNo 77.0 (295) 71.1 (108)Married/partneredYes 47.4 (182)52.0 (79)NSNo 52.6 (202) 48.0 (73)EmployedYes 51.4 (197)49.7 (76)NSNo 48.6 (186) 50.3 (77)HandednessRight 88.8 (341)88.9 (136)Left 8.1 (31)9.2 (14)NSBoth 3.1 (12)2.0 (3)Occurrence of comorbid conditions (% and number of womenwho reported each comorbid condition from theSelf-Administered Comorbidity Questionnaire)Heart disease5.6 (21)6.0 (9)NSHigh blood pressure27.0 (103) 34.9 (53)NSLung disease3.7 (14) 8.1 (12)p=.04Diabetes 6.6 (25) 7.4 (11)NSUlcer3.7 (14) 4.7 (7)NSKidney disease1.6 (6)2.0 (3) NSLiver disease2.1 (8) 4.8 (7)NSAnemia 7.2 (27)9.5 (14) NSDepression21.8 (81) 26.7 (39)NSOsteoarthritis 19.2 (72)26.7 (40)NSBack pain29.3 (110)31.5 (47)NSRheumatoid arthritis3.5 (13) 4.7 (7) NSDiagnosed with mastitisYes 13.1 (50) 11.3 (17) NSNo86.9 (332) 88.7 (134)Diagnosed with cystic breast diseaseYes 21.5 (81)23.3 (34) NSCandidate Genes and LymphedemaPLOS ONE | www.plosone.org9 April 2013 | Volume 8 | Issue 4 | e60164
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Table 2. Cont.% (n)% (n)No 78.5 (295)76.7 (112)BreastfedYes49.4 (190)45.1 (69)NSNo50.6 (195) 54.9 (84)Surgery on affected breast not related to cancerYes 9.3 (36)14.8 (23) NSNo90.7 (351) 85.2 (132)Surgery to the affected arm not related to cancerYes3.1 (12) 5.2 (8) NSNo96.9 (375) 94.8 (147)Surgery on the affected hand not related to cancerYes5.2 (20)7.1 (11)NSNo 94.8 (367) 92.9 (144)Injury to the affected armYes 17.3 (67)22.6 (35) NSNo 82.7 (320)77.4 (120)Injury to the affected handYes 17.1 (66)17.4 (27)NSNo 82.9 (321)82.6 (128)Side of cancer surgeryDominant49.9 (193)41.9 (65) NSNondominant50.1 (194) 58.1 (90)Type of surgeryBreast conservation75.2 (291)70.3 (109) NSMastectomy24.8 (96) 29.7 (46)Stage of diseaseStage 018.1 (70) 5.2 (8)Stage I40.1 (155)32.9 (51)p,.0001Stage IIA and IIB35.4 (137) 48.4 (75)Stage IIIA, IIIB, IIIC, and IV6.5 (25)13.5 (21)Sentinel lymph node biopsyYes 80.9 (313)69.7 (108)p=.006No 19.1 (74)30.3 (47)Axillary lymph node dissectionYes 39.3 (152) 69.3 (106)p,.0001No 60.7 (235) 30.7 (47)Reconstruction at the time of surgeryYes 21.6 (68) 22.2 (18) NSNo 78.4 (247) 77.8 (63)Adjuvant chemotherapyYes 36.7 (142) 59.7 (92)p,.0001No 63.3 (245)40.3 (62)Adjuvant radiation therapyYes57.1 (221)71.0 (110)p,.0001No 42.9 (166) 29.0 (45)Combinations of treatmentsOnly surgery 23.8 (92) 8.4 (13)Surgery and radiation therapy39.5 (153)32.3 (50)p,.0001Surgery and chemotherapy19.1 (74)20.6 (32)Candidate Genes and LymphedemaPLOS ONE | www.plosone.org10 April 2013 | Volume 8 | Issue 4 | e60164
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SYK: 19 SNPs; VCAM1: 7 SNPs; VEGFB: 1 SNP; VEGFC: 8SNPs; VEGFD: 4 SNPs; VEGFR2: 12 SNPs, VEGFR3: 8 SNPs)passed all quality control filters and were included in the geneticassociation analyses. The one SNP used to evaluate geneticvariation in EMILIN1 did not pass the quality control filters.Potential functional roles of SNPs associated with LE wereexamined using PUPASuite 2.0 [32], a comprehensive searchengine that predicts a series of functional effects (i.e., non-synonymous changes, altered transcription factor binding sites,exonic splicing enhancing or silencing, splice site alterations,microRNA target alterations).Statistical Analyses.Allele and genotype frequencies weredetermined by gene counting. Hardy-Weinberg equilibrium wasassessedbytheChi-squareorFisherExacttests.Measuresoflinkagedisequilibrium ((LD) i.e., D' and r2) were computed from theparticipants' genotypes with Haploview 4.2. LD-based haplotypeblock definition was based on D' confidence interval [33].For SNPs that were members of the same haploblock, haplotypeanalyses were conducted in order to localize the association signalTable 2. Cont.% (n) % (n)Surgery, radiation therapy, and chemotherapy17.6 (68)38.7 (60)Exercise on a regular basisYes73.7 (283)75.2 (115) NSNo26.3 (101)24.8 (38)Abbreviations: kg = kilograms, m2– meter squared, NS = not significant, SD = standard deviation.doi:10.1371/journal.pone.0060164.t002Table 3. Multiple logistic regression analyses for phenotypic predictors of the development of lymphedema.Regression analysis WITHOUT the inclusion of genomic and self-reported race/ethnicityPredictor Odds Ratio Standard Error95% CIZ p-valueBMI 1.060.018 1.025, 1.097 3.390.001Stage of diseaseStage 0 versus IStage 0 versus IIStage 0 versus III and IV22.214.171.1241.5901.6222.1511.234, 8.4791.171, 8.6401.129, 11.6032.392.272.160.0170.0230.030SLNB0.42 0.1160.243, 0.71923.16 0.002Number of nodes removed1.06 0.0171.028, 1.093 3.70,.0001Any chemotherapy 1.74 0.4161.086, 2.779 2.30 0.021Any radiation therapy 1.940.452 1.224, 3.060 2.830.005Overall model fit: x2=85.32, p,0.0001, R2=0.1380Regression analysis WITH the inclusion of genomic and self-reported race/ethnicityBMI 1.05 0.0221.012, 1.0992.56 0.010Stage of diseaseStage 0 versus IStage 0 versus IIStage 0 versus III and IV3.003.765.841.7732.3594.2570.943, 9.5541.102, 12.8581.399, 24.3721.862.122.420.0630.0340.015SLNB 0.40 0.1370.206, 0.78422.670.007Number of nodes removed1.08 0.0211.043, 1.126 4.10,.0001Any chemotherapy 1.200.368 0.654, 2.184 0.58 0.561Any radiation therapy 1.32 0.3900.738, 2.356 0.930.351Self-reported race/ethnicityWhite versus BlackWhite versus Asian/Pacific IslanderWhite versus Hispanic/Mixed4.352.902.354.8802.6931.2900.483, 39.2010.472, 17.8750.798, 6.8901.311.151.550.1900.2500.121Genomic race/ethnicityAIMS – Principalcomponent 1 AIMS – Principalcomponent 2 AIMS – Principalcomponent 30.750.861.100.1210.1040.1220.549, 1.0300.678, 1.0900.886, 1.36721.7821.250.870.0760.2110.386Overall model fit: x2=77.83, p,0.0001, R2=0.1735Abbreviations: AIMS = ancestry informative markers; BMI = body mass index; CI =confidence interval; SLNB = sentinel lymph node biopsy.doi:10.1371/journal.pone.0060164.t003Candidate Genes and LymphedemaPLOS ONE | www.plosone.org11 April 2013 | Volume 8 | Issue 4 | e60164
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Table 4. Multiple logistic regression analyses for FOXC2. LCP2, NRP2, SYK, VCAM1, and VEGF-C genotypes and halotypes to predictthe development of lymphedema.Predictor Odds Ratio Standard Error 95% CIZ p-valueFOXC2 haplotype 0.630.125 0.430, 0.93122.32 0.020BMI1.060.022 1.016, 1.1042.72 0.006Stage of diseaseStage 0 versus IStage 0 versus IIStage 0 versus III and IV3.434.106.712.0532.6194.9751.060, 11.0851.175, 14.3331.571, 28.6872.062.212.570.0400.0270.010SLNB 0.40 0.1380.203, 0.78422.67 0.008Number of nodesremoved1.080.022 1.042, 1.1274.01,.0001Any chemotherapy1.22 0.3800.664, 2.246 0.64 0.521Any radiation therapy 1.35 0.4060.752, 2.436 1.010.313Overall model fit: x2=83.44, p,0.0001, R2=0.1860LCP2 genotype 0.500.132 0.298, 0.83822.630.009BMI 1.050.022 1.012, 1.0972.54 0.011Stage of diseaseStage 0 versus IStage 0 versus IIStage 0 versus III and IV3.013.565.941.7882.2384.3650.937, 9.6431.040, 12.2031.407, 25.0791.852.022.420.0640.0430.015SLNB0.40 0.1370.204, 0.78422.67 0.008Number of nodesremoved1.08 0.022 1.040, 1.1253.93,.0001Any chemotherapy 1.25 0.3890.679, 2.298 0.710.475Any radiation therapy1.33 0.398 0.744, 2.3920.970.334Overall model fit: x2=84.86, p,0.0001, R2=0.1891NRP2 genotype0.38 0.143 0.185, 0.79922.56 0.010BMI 1.06 0.0231.016, 1.105 2.680.007Stage of diseaseStage 0 versus IStage 0 versus IIStage 0 versus III and IV2.793.416.011.6472.1504.4300.874, 8.8750.990, 11.7331.420, 25.4771.731.942.440.0830.0520.015SLNB0.390.136 0.203, 0.77522.71 0.007Number of nodesremoved1.080.021 1.040, 1.122 4.00,0.0001Any chemotherapy1.18 0.369 0.635, 2.1740.51 0.607Any radiation therapy 1.200.3630.667, 2.171 0.61 0.539Overall model fit: x2=85.11, p,0.0001, R2=0.1897NRP2 haplotype 0.540.114 0.358, 0.81722.920.003BMI1.050.022 1.011, 1.099 2.480.013Stage of diseaseStage 0 versus IStage 0 versus IIStage 0 versus III and IV3.033.815.491.8052.4334.0810.941, 9.7381.088, 13.3211.281, 23.5611.862.092.290.0630.0360.022SLNB0.40 0.137 0.201, 0.78022.68 0.007Number of nodes removed 1.090.021 1.046, 1.1324.21,0.0001Any chemotherapy 1.110.3480.602, 2.0540.34 0.734Any radiation therapy 1.300.3910.723, 2.346 0.880.379Overall model fit: x2=86.91, p,0.0001, R2=0.1937SYK genotype3.43 1.1311.797, 6.5463.74,0.0001BMI1.06 0.023 1.017, 1.1062.76 0.006Stage of diseaseStage 0 versus IStage 0 versus IIStage 0 versus III and IV3.053.105.501.8211.9684.0780.946, 9.8320.891, 10.7601.286, 23.5231.871.782.300.0620.0750.022SLNB 0.400.141 0.201, 0.79722.61 0.009Candidate Genes and LymphedemaPLOS ONE | www.plosone.org12April 2013 | Volume 8 | Issue 4 | e60164
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within each gene and to determine if haplotypes improved thestrength of the association with the phenotype. Haplotypes wereconstructed using the program PHASE version 2.1 [34]. In orderto improve the stability of haplotype inference, the haplotypeconstruction procedure was repeated five times using different seednumbers with each cycle. Only haplotypes that were inferred withprobability estimates of $.85, across the five iterations, wereretained for downstream analyses. Haplotypes were evaluatedassuming a dosage model (i.e., analogous to the additive model).Ancestry informative markers (AIMS) were used to minimizeconfounding due to population stratification [35–37]. Homoge-neity in ancestry among participants was verified by principalcomponent analysis [38], using Helix Tree (Golden Helix,Bozeman, MT). Briefly, the number of principal components(PCs) was sought which distinguished the major racial/ethnicgroups in the sample by visual inspection of scatter plots oforthogonal PCs (i.e., PC 1 versus PC2, PC2 versus PC3). Thisprocedure was repeated until no discernible clustering of patientsby their self-reported race/ethnicity was possible (data not shown).One hundred and six AIMs were included in the analysis. The firstthree PCs were selected to adjust for potential confounding due topopulation substructure (i.e., race/ethnicity) by including the threecovariates in all regression models.For association tests, three genetic models were assessed for eachSNP: additive, dominant, and recessive. Barring trivial improve-ments (i.e., delta ,10%), the genetic model that best fit the data,by maximizing the significance of the p-value, was selected foreach SNP. Logistic regression analysis, that controlled forsignificant covariates, as well as genomic estimates of and self-reported race/ethnicity, was used to evaluate the associationbetween genotype and LE group membership. A backwardsstepwise approach was used to create the most parsimoniousmodel. Genetic model fit and both unadjusted and covariate-adjusted odds ratios were estimated using STATA version 9 [39].As was done in our previous studies [40–42], based onrecommendations in the literature [43,44], the implementationTable 4. Cont.PredictorOdds Ratio Standard Error95% CIZ p-valueNumber of nodesremoved1.10 0.0221.054, 1.142 4.53,0.0001Any chemotherapy1.44 0.4590.773, 2.6921.15 0.249Any radiation therapy1.45 0.4370.803, 2.616 1.23 0.218Overall model fit: x2=91.90, p,0.0001, R2=0.2049VCAM1 genotype0.550.158 0.309, 0.96322.090.037BMI 1.06 0.0221.015, 1.102 2.680.007Stage of diseaseStage 0 versus IStage 0 versus IIStage 0 versus III and IV2.853.785.921.7102.4134.3850.879, 9.2391.080, 13.2141.388, 25.2751.752.082.400.0810.0380.016SLNB 0.410.1410.206, 0.80122.600.009Number of nodesremoved1.080.022 1.043, 1.128 4.07,0.0001Any chemotherapy1.18 0.365 0.643, 2.161 0.53 0.596Any radiation therapy 1.30 0.3870.724, 2.327 0.880.381Overall model fit: x2=82.35, p,0.0001, R2=0.1836VEGFC haplotype 0.64 0.1450.409, 0.99721.970.049BMI 1.05 0.022 1.011, 1.097 2.46 0.014Stage of diseaseStage 0 versus IStage 0 versus IIStage 0 versus III and IV2.943.745.421.7402.3513.9620.920, 9.3801.091, 12.8201.291, 22.7161.822.102.310.0690.0360.021SLNB0.420.1450.215, 0.82622.510.012Number of nodesremoved1.09 0.021 1.044, 1.1284.16,0.0001Any chemotherapy 1.240.387 0.675, 2.287 0.700.487Any radiation therapy 1.31 0.391 0.729, 2.354 0.900.366Overall model fit: x2=81.85, p,0.0001, R2=0.1825For each model, the first three principal components identified from the analysis of ancestry informative markers as well as self-report race/ethnicity (i.e., White, Black,Asian/Pacific Islander, Hispanic/Mixed ethnic background/Other) were retained in all models to adjust for potential confounding due to race or ethnicity (data notshown). Predictors evaluated in each model included genotype (FOXC2 haplotype A03 composed of the rs34221221 ''C'' allele and the rs1035550 ''C'' allele; LCP2rs315721: AA versus AG+GG; NRP2 rs849530: TT+TG versus GG; NPR2 haplotype F01 composed of the rs849530 ''G'' allele, the rs950219 ''G'' allele, and the rs3771052 ''G''allele; SYK rs158689: AA+AT versus TT; VCAM1 rs3176861: CC versus CT + TT; VEGFC haplotype B03 composed of the rs3775202 ''G'' allele and the rs3775195 ''C'' allele),BMI (kilograms/meter squared), stage of disease, SLNB, number of lymph nodes removed, receipt of chemotherapy prior to or following surgery, and receipt of radiationtherapy following surgery.Abbreviations: BMI = body mass index; CI =confidence interval; FOXC2 = Forkhead box protein C2; LCP2 = Lymphocyte cytosolic protein 2; NRP = neuropilin-2;SLNB = sentinel lymph node biopsy; SYK = spleen tyrosine kinase; VCAM1 = vascular cell adhesion molecule 1; VEGFC = vascular endothelial growth factor-C.doi:10.1371/journal.pone.0060164.t004Candidate Genes and LymphedemaPLOS ONE | www.plosone.org 13 April 2013 | Volume 8 | Issue 4 | e60164
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of rigorous quality controls for genomic data, the non-indepen-dence of SNPs/haplotypes in LD, and the exploratory nature ofthe analyses, adjustments were not made for multiple testing. Inaddition, significant SNPs identified in the bivariate analyses wereevaluated further using regression analyses that controlled fordifferences in phenotypic characteristics, potential confoundingdue to population stratification, and variation in other SNPs/haplotypes within the same gene. Only those SNPs that remainedsignificant were included in the final presentation of the results.Therefore, the significant independent associations reported areunlikely to be due solely to chance. Unadjusted (bivariate)associations are reported for all SNPs passing quality controlcriteria in Table 1 to allow for subsequent comparisons and meta-analyses.ResultsDifferences in demographic and clinical characteristicsAs shown in Table 2, no differences were found betweenpatients with and without LE for the majority of the demographicand clinical characteristics. Patients with LE had a significantlyhigher BMI and a lower KPS score, and were more likely to reportlung disease. In addition, patients with LE had a higher number oflymph nodes removed, a higher number of positive nodes, moreadvanced disease at the time of diagnosis, were less likely to havehad a SLNB, and were more likely to have had an ALND, hadreceived CTX prior to or following surgery, and had received RTfollowing surgery.Regression analyses for phenotypic predictors of LEAs shown in Table 3, two regression analyses were done toevaluate the associations between phenotypic characteristics andLE group membership. In the first regression analysis, thatincluded BMI, stage of disease, SLNB status, number of lymphnodes removed, receipt of CTX, and receipt of RT predicted13.8% of the variance in LE group membership (p,0.0001). Theodds of developing LE increased significantly for women who hada higher BMI, had a higher stage of disease at diagnosis, had ahigher number of lymph nodes removed, had received CTX priorto or following surgery, and had received RT. The odds of havingLE decrease significantly in women who had a SLNB.In the second regression analysis that added genomic estimatesof and self-reported race/ethnicity in addition to the character-istics listed above, the overall model explained 17.4% of thevariance in LE group membership (p,0.0001). While BMI, stageof disease, SLNB status, and number of lymph nodes removedremained significant, when genomic and self-reported race/ethnicity were included in the model, the receipt of CTX andRT were no longer significant predictors of LE group member-ship.Candidate gene analyses for the development of LEAs summarized in Table 1, no associations with the occurrenceof LE were found in the SNPs evaluated for HGF, LYVE1, MET,PROX1, RORC, VEGFB, VEGFD, and VEGFR3. However, thegenotype frequency was significantly different between those whodid and did not develop LE for thirteen SNPs and sevenhaplotypes spanning nine genes (i.e., ANGPT2, FOXC2, LCP2,NRP2, SOX 17, SYK, VCAM1, VEGFC, and VEGFR2). For theSNP in ANGPT2 (rs6990020), a dominant model fit the data best(p=.040). One haplotype (HapA03, p=.045) was identified inFOXC2. For the three SNPs (rs572192, rs315721, rs6866733)identified in LCP2, a dominant model fit the data best (p=.047,p=.005, p=.026, respectively). Three SNPs (rs849530, rs849563,rs16837641) and one haplotype (HapF01, p=.011) were identifiedin NRP2. For all three SNPs, a recessive model fit the data best(p=.042, p=.012, p=.009, respectively). For the SNP in SOX17(rs12541742), a dominant model fit the data best (p=.008). OneSNP (rs158689) and 1 haplotype (HapD01, p=.008) wereidentified in SYK. For rs158689, a recessive model fit the databest (p=.039). One SNP (rs3176861) and one haplotype (HapB01,p=.016) were identified in VCAM1. For rs3176861, a dominantmodel fit the data best (p=.036). One SNP (rs7664413) and twohaplotypes (HapA05, p=.046; HapB03, p=.027) were identifiedin VEGFC. For rs7664413, an additive model fit the data best(p=.041). Two SNPs (rs10020464, rs11133360) and one haplo-type (HapC04, p=.021) were identified for VEGFR2. Forrs10020464, a dominant model fit the data best (p=.025) andfor rs11133360 a recessive model fit the data best (p=.032).Regression analyses of FOXC2, LCP2, NRP2, SYK, VCAM1,and VECFC genotypes and haplotypes and thedevelopment of LEIn order to better estimate the magnitude (i.e., odds ratio, OR)and precision (95% confidence interval, CI) of genotype on thedevelopment of LE, multivariate logistic regression models werefit. As shown in Table 4, in addition to genotype, the phenotypicvariables included in the regression models were ethnicity (i.e.,White, Black, Asian, Hispanic/Mixed ethnic background/Other),Figure 1. FOXC2 Gene Structure and Linkage Disequilibrium.An ideogram of forkhead box C2 (FOXC2) is presented above the whitebar that represents the physical distance along human chromosome 16(chr16: 85,158,358–85,160,040; genome assembly 36.3, NM_005251.2).Exons are represented as thick bars. Reference sequence identifiers(rsID) for each single nucleotide polymorphism (SNP) are plotted bothin terms of their physical distance (i.e., the white bar at the top of thefigure) and equidistantly to render the pairwise linkage disequilibrium(LD) estimates that were calculated and visualized with Haploview 4.2.The gene structure for FOXC2 was rendered with FancyGene 1.4. Thecorrelation statistic (r2and D') is provided in the heatmap. LD-basedhaplotype block definition was based on the D' confidence intervalmethod. The haploblock is indicated in a bolded triangle and itscomponent SNPs are rendered in bold font. Pairwise D' value (range: 0–1, inclusive) was rendered in color, with darker red diamondrepresenting D' value approaching 1.0. When the r2value (range of0–100, inclusive) is not equal to 0 or 100, it is provided in a givendiamond. The 2-SNP haplotype associated with LE is composed of onerare and one common allele of two SNPs located in the immediate earlypromoter (rs34221221; rare ''C'' allele) and immediately downstream ofthe FOXC2 coding region (rs1035550; common ''C'' allele).doi:10.1371/journal.pone.0060164.g001Candidate Genes and LymphedemaPLOS ONE | www.plosone.org14 April 2013 | Volume 8 | Issue 4 | e60164
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BMI, stage of disease, having a SLNB, and number of lymphnodes removed. Receipt of CTX and RT, while not significantafter the inclusion of genomic estimates of and self-reported race/ethnicity, were retained in all of the regression models for facevalidity.The only genetic associations that remained significant in themultivariate logistic regression analyses were for FOXC2 haplo-type A03, LCP2 rs315721, NRP2 rs849530, NRP2 haplotype F01,SYK rs158689, VCAM1 rs3176861, and VEGFC haplotype B03(see Table 4 and Figures 1, 2, 3, and 4). In the regression analysisfor the FOXC2 haplotype A3, which was composed of two SNPs(i.e., rs34221221, rs1035550), the overall model explained 18.6%of the variance in the odds of developing LE. Each additional doseof the FOXC2 haplotype A03 was associated with 37.0% decreasein the odds of developing LE. Figure 1 displays the FOXC2 LD-based heatmap and haplotype analysis.In the regression analysis for LCP2 rs315721, the overall modelexplained 18.9% of the variance in the odds of developing LE.Carrying one or two doses of the rare allele (i.e., AA versus AG +GG) was associated with a 50.0% decrease in the odds ofdeveloping LE (Figure 2A).In the regression analysis for NRP2 rs849530, the overall modelexplained 19.0% of the variance in the odds of developing LE.Carrying two doses of the rare allele (i.e., TT+TG versus GG) wasassociated with 62.0% decrease in the odds of developing LE(Figure 2B). In the regression analysis for the NRP2 haplotypeF01, which was composed of three SNPs (i.e., rs849530, rs950219,rs3771052), the overall model explained 19.4% of the variance inthe odds of developing LE. Each additional dose of the NRP2haplotype F01 was associated with 46.0% decrease in the odds ofdeveloping LE. Figure 3 displays the NRP2 LD-based heatmapand haplotype analysis.In the regression analysis for SYK rs158689, the overall modelexplained 20.1% of the variance in the odds of developing LE.Carrying two doses of the rare allele (i.e., AA + AT versus TT) wasassociated with 3.43-fold increase in the odds of developing LE(Figure 2C).In the regression analysis for VCAM1 rs3176861, the overallmodel explained 18.4% of the variance in the odds of developingLE. Carrying one or two doses of the rare allele (i.e., CC versusCT + TT) was associated with a 45.0% decrease in the odds ofdeveloping LE (Figure 2D).Figure 2. Differences between the lymphedema and no lymphedema groups. A – Differences between the lymphedema and nolymphedema groups in the percentages of patients who were homozygous for the common allele (AA) or heterozygous or homozygous for the rareallele (AG+GG) for rs315721 in lymphocyte cytosolic protein 2 (LCP2). B – Differences between the lymphedema and no lymphedema groups in thepercentages of patients who were homozygous or heterozygous for the common allele (TT+TG) or homozygous for the rare allele (GG) for rs849530in neuropilin-2 (NRP2). C – Differences between the lymphedema and no lymphedema groups in the percentages of patients who were homozygousor heterozygous for the common allele (AA+AT) or homozygous for the rare allele (TT) for rs158689 in protein tyrosine kinase (SYK). D – Differencesbetween the lymphedema and no lymphedema groups in the percentages of patients who were homozygous for the common allele (CC) orheterozygous or homozygous for the rare allele (CT+TT) for rs3176861 in vascular cell adhesion molecule 1 (VCAM1).doi:10.1371/journal.pone.0060164.g002Candidate Genes and LymphedemaPLOS ONE | www.plosone.org 15 April 2013 | Volume 8 | Issue 4 | e60164
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